Methicillin resistant strains of Staphylococcus aureus (MRSA) are implicated in infections with serious outcomes including nosocomial outbreaks, and show resistance to a wide range of antibiotics, thus limiting the treatment options. Healthcare associated MRSA is of particular clinical importance because it is not only predictably cross resistant to all penicillins and cephalosporins, but is also typically resistant to multiple other commonly used antibiotics. Treatment of MRSA infections generally require more expensive and often more toxic antibiotics, which are normally used as the last line of defense. Therefore, rapid detection of MRSA is clinically crucial for both treatment and infection control measures.
Detection of MRSA is further complicated by the fact that MRSA can often co-colonize with multiple other related bacteria, including methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant coagulase-negative staphylococci (MR-CoNS) and/or methicillin-sensitive coagulase-negative staphylococci (MS-CoNS).
Traditional methods for the detection of MRSA in clinical microbiology laboratories involve culturing the bacteria from a sample as the first step for the isolation and differentiation of MRSA from MSSA and MR-CoNS. This approach is time-consuming and requires a minimum of 20 to 24 hours until a result is known.
A number of molecular based methods have been published for the detection of methicillin resistant Staphylococcus aureus (MRSA) and differentiating it from methicillin sensitive Staphylococcus aureus (MSSA). One such method targets two separate regions of MRSA, the mecA gene of the Staphylococcus cassette chromosome (SCCmec, responsible for methicillin resistance) and spa gene of Staphylococcus aureus (U.S. Pat. No. 5,702,895, Sinsimer, et al., Journal of Clinical Microbiology, September 2005, 4585-4591). Unambiguous detection of MRSA using this approach is hampered by the co-existence of non-Staphylococcus aureus strains such as methicillin resistant coagulase negative Staphylococci (MR-CoNS) which also harbors the mecA gene for methicillin resistance (Becker, et. al. Journal of Clinical Microbiology, January 2006, p 229-231).
A more recent molecular approach utilizes primers and probes to SCCmec and the flanking region of the Staphylococcus aureus genome (U.S. Pat. No. 6,156,507, Huletsky, et. al. Journal of Clinical Microbiology, May 2004, p 1875-1884). SCCmec is a mobile genetic element that carries the mecA gene and inserts at a specific site, attBscc, at the 3′-end of the orfX gene. The left extremity of SCCmec is contiguous with the non-orfX side of attBscc while the right extremity of SCCmec is contiguous with the orfX side of attBscc (Ito, et al., Antimicrob. Agent Chemother. 2001, 45, p 1323-1336; Ito et al., Antimicrob. Agent Chemother. 2004, 48, p 2637-2651, Noto, et al., J. Bacteriol. 2008, 190:1276-1283). This approach infers the presence of the mecA gene from the detection of the SCCmec/orfX junction. This approach requires the use of multiple primers as there have been several different types of SCCmec described. This approach is also subject to false positive results due to the presence of SCCmec cassettes that do not contain the mecA gene (Farley, et. al. Journal of Clinical Microbiology, February 2008, p 743-746) and false negative results due to newly emerged SCCmec types not covered by the assay (Heusser, et al., Antimicrob. Agents Chemother. January 2007, p 390-393).
Another approach utilizes one primer in a region of high homology across the different SCCmec types and one primer in the flanking Staphylococcus aureus DNA (Cuny, et al. Clin. Microbiol Infect 2005; 11:834-837, European Patent 1529847 B1). This approach is also subject to false positives as the probability of also priming of MSSA is high with primers encompassing this region.
Finally, a method has been described that positively selects for Staphylococcus aureus using specific antibodies and magnetic beads (Francois, et al. Journal of Clinical Microbiology, January 2003, p 254-260; European Patent 1,370,694B1). This approach enriches for Staphylococcus aureus, but requires the use of three primer/probe sets to positively identify MRSA and reduces the possibility of detecting CoNS. The method requires a centrifugation step and a separate lysis step to recover the nucleic acid.
The commercially available MRSA assays target the SCCmec right extremity junction and orfX. Five different types and numerous subtypes of SCCmec have been identified and the potential of emergence of new SCCmec subtypes is high. In addition, it is possible that MSSA derived from MRSA might retain part of the SCCmec sequence without the mecA gene. Therefore, assays targeting the SCCmec right extreme junction with orfX are likely to give false positive results with MRSA-derived MSSA and false negative results with MRSA carrying newly emergent SCCmec types/subtypes.
Thus, current methods for detection of MRSA are laborious, time-consuming, and unreliable for routine clinical and surveillance purposes. Accordingly, there exists a need for developing an assay that is fast, easy, reliable and capable of detecting and concurrently distinguishing MRSA from other related bacteria, including MSSA, and/or MR-CoNS.